This application is filing under 35 U.S.C. xc2xa7371 of International Patent Application No. PCT/EP97/04215, filed on Aug. 1, 1997, which in turn is an international filing of German Patent Application No. 19632862.4, filed on Aug. 14, 1996.
Lyme borreliosis is the most frequent of the infectious diseases of humans which are transmitted by ticks. A substantial proportion of the ticks which serve as vectors for transmitting Lyme borreliosis are infected with the pathogen of Lyme borreliosis, i.e. the spirochete Borrelia burgdorferi. Depending on the geographic region, the percentage infected can vary from 1% up to 100%.
An infection with B.burgdorferi leads to a complex clinical picture which can be subdivided into different stages.
In some cases, the infection with B.burgdorferi can take a subclinical course. However, late sequelae, which are caused by unrecognized or untreated Borrelia infections, frequently present a problem. Particularly because of the dangerous diseases, such as carditis, myositis, iritis, panophthalmitis or neurological manifestations, which can occur when infections are not recognized or not treated, it is important to be able to diagnose a possible infection with B.burgdorferi as reliably and accurately as possible.
The pathogen can be detected in patient material, in particular in the early stages. However, it is disadvantageous in this context that culturing B.burgdorferi is relatively difficult and therefore as a rule left to specialist laboratories.
It is desirable, therefore, to detect the antibodies in serum and also in cerebrospinal fluid, in the case of neurological manifestations, and in joint aspirates in the case of joint ailments.
For diagnosis, it is important to be able to decide whether an infection only occurred recently or whether the infection is one which took place some while ago. This distinction can be made by immunologically determining the nature of the detected antibodies; as a rule, IgM antibodies suggest that the infection only occurred recently whereas IgG antibodies suggest that the infection took place some while ago.
It is also important for. diagnosis that the diagnostic tests are specific, i.e. that no cross-reactions occur with those bacterial pathogens, such as Treponema pallidum, which are to a certain degree phylogenetically related to the borrelias.
On the other hand, however, it is also of importance for diagnosis that, if at all possible, all the strains of Borrelia burgdorferi can be recognized by the proteins or peptides which are employed in the test method.
Since Lyme borreliosis is widespread and since an infection can readily be transmitted by means of a tick bite, there is also a substantial need to develop vaccines which ensure immune protection against borrelia infections.
Those proteins which, on the surface of the bacteria, come into contact with the immune system of the infected organism are particularly suitable for developing vaccines.
Two proteins have now been found, within the context of the present invention, which are particularly suitable both for diagnosis and for developing vaccines.
The present invention relates, therefore, to immunologically active proteins from Borrelia burgdorferi which are present in a form which is free of other proteins derived from Borrelia burgdorferi and which exhibit the sequence of the protein 1829-22A, having the amino acid sequence (SEQ ID NO:1)
MKKFNLIIEALFAILLTACNFGLMEETKIALESSSKDVKNKILQIKKDAEDKGVNFAAFTSSETGSKVTNGGLALREAKIQAINEVEKFLKRIEEEALKLKEHGNSGQFLELFDLLLEVLESLEPIGIKGLKDFISEEAKCNPISTSERLIEVKVQIENKMEEVKRKQNLNKERKSNKGKKKK
or a part sequence thereof having at least 10 consecutive amino acids, or a part sequence therof having at least 10 consecutive amino acids, or the sequence of the protein 1829-22B, having the amino acid sequence (SEQ ID NO:2)
MIKYNKIILTLTLLASLLAACSLTGKARLESSVKDITNEIEKAIKEAEDAGVKTDAFTETQTGGKVAGPKIRAAKIRVADLTIKFLEATEEETITFKENGAGEDEFSGIYDLILNAAKAVEKIGMKDMTKTVEEAAKENPKTTANGIIEIVKVMKAKVENIKEKQTKNQK
or a part sequence thereof having at least 10 consecutive amino acids.
In accordance with the invention, preference is given to using those part sequences which possess epitopes which are diagnostically and/or therapeutically relevant. In the case of protein 1829-22A, whose sequence is given in the sequence listing under Seq. ID No. 1, the following part sequences are particularly preferred: The region between amino acid 31 (Lys) and amino acid 55 (Asn). Another preferred polypeptide is located between position 60 (Thr) and position 71 (Gly). A further preferred polypeptide is located between amino acid 82 (Gln) and amino acid 108 (Gln). The C-terminal region between amino acid 130 (Gly) and amino acid 183 (Lys) is also particularly preferred.
In the case of protein 1829-22B, which is represented by Seq. ID No. 2, the following part regions are particularly preferred: amino acid 61 (Gln) to amino acid 71 (Ile); amino acid 87 (Glu) to amino acid 108 (Gly); amino acid 121 (Glu) to amino acid 145 (Asn), and the C-terminal region from amino acid 150 (Ile) to amino acid 170 (Lys). The positions of the amino acids are given in the sequence listings. The peptides which exhibit the abovementioned part sequences can either be prepared by means of chemical synthesis or be expressed recombinantly in suitable host systems.
The proteins or peptides according to the invention may be prepared by means of recombinant methods, which has the advantage that no other proteins derived from B.burgdorferi are associated with the desired proteins. Alternatively, suitable peptides may also be synthesized in the classical chemical manner; such peptides are also free of immunologically inactive impurities. However, it is entirely possible to employ the proteins or peptides according to the invention in test kits or in vaccines together with other proteins which have been isolated from B.burgdorferi. 
The term xe2x80x9cimmunologically active proteinxe2x80x9d, which is used within the context of the present invention, encompasses not only a protein which comprises the complete amino acid sequence of protein 1829-22A or protein 1829-22B but also parts of these proteins which are at least long enough to encompass at least one linear epitope. In general, the minimum length of a peptide according to the invention which is able to exhibit the property of an epitope is at least 6, preferably 10, particularly preferably 25 and very particularly preferably at least 50 amino acids.
The fact must be taken into consideration that, in the individual strains of Borrelia burgdorferi, at least minor changes occur in the amino acid sequence of the protein, depending on the particular strain. The present invention therefore also relates to immunologically active proteins or peptides which exhibit a high degree of homology with the above-described amino acid sequences.
The immunologically active proteins or peptides according to the invention exhibit an homology of at least 60%, preferably at least 80% and particularly preferably at least 90%, based on proteins 1829-22A and 1829-22B according to the invention. The term an homology of 90% is understood as meaning, for example, that, in the homologous peptide, 9 out of 10 amino acids are identical to the corresponding amino acids at the homologous sites in amino acid sequence 1829-22A or amino acid sequence 1829-22B.
Within the context of the present invention, those regions of the proteins or peptides according to the invention are particularly important which exhibit epitopes, that is sites in the protein to which antibodies bind specifically. Determining at which sites epitopes are to be expected can either be achieved using computer methods which are known per se, or it is also possible to synthesize defined short peptides having a length of at least 10, preferably at least 25, amino acids. These peptides are then tested with positive sera to determine whether immunological reactions do or do not take place. In this way, it is possible to identify linear epitopes. When preparing these proteins or peptides, either use can be made of recombinant methods, with the peptides, for example, being expressed in microorganisms as fusion proteins, or the peptides can be synthesized by means of classical synthesis (Merrifield technique).
Identifying immunologically relevant epitopes is important not only for diagnosis but also, in particular, for preparing vaccines. For vaccines, regions of the proteins according to the invention which are very reactive immunologically can be combined with appropriate regions of other, previously known proteins from Borrelia burgdorferi, such as OspA or OspC, or with flagellin amino acid sequences.
The present invention also relates to test kits for detecting antibodies against Borrelia strains, which test kits contain at least one immunologically active protein according to the invention, which is able to react with the antibodies which are present in the fluid under investigation, and which contain at least one reporter component which makes it possible to detect complexes consisting of immunologically active protein and antibody.
Preference is given to test kits which contain at least one immunologically active protein having a part sequence of protein 1829-22A and at least one protein having a part sequence of protein 1829-22B.
The reporter component can be an antibody which is directed against the antibody to be detected and which exhibits a label. In this context, the reporter component is preferably an anti-human IgG antibody or an anti-human IgM antibody. The label is frequently an enzyme which is able to catalyze a color reaction.
One detection possibility consists in the immunologically active protein according to the invention, or a monoclonal antibody which is directed against it, being biotinylated and the reporter component being avidin or streptavidin to which an enzyme, in particular peroxidase, is covalently bonded.
In a preferred embodiment of the invention, the test kit is an ELISA test kit. In a particularly preferred embodiment of the present invention, at least one immunologically active protein according to the invention is coupled to microtiter plates, and the reporter component consists of anti-human immunoglobulin, in particular anti-IgG antibodies and/or anti-IgM antibodies, to which an enzyme which catalyzes a color reaction is coupled.
In another preferred embodiment of the present invention, the test kit is an immunoblot, which is also described as a protein blot or a western blot. In test kits of this nature, protein is transferred, using an electrophoresis gel, for example a polyacrylamide gel, onto an immobilizing matrix (e.g. nitrocellulose filter). The transfer can be effected, for example, by means of electrotransfer. An immunological reaction then takes place between the proteins present on the matrix and the antibodies which are directed against the proteins. The antibodies can then be detected by means of suitable methods, e.g. using enzyme-labeled anti-antibody antibodies.
The term xe2x80x9ctest kitsxe2x80x9d is understood as meaning a set of test reagents which makes it possible to detect particular antibodies. The test kits according to the invention contain, as the component according to the invention, at least one protein or peptide according to the invention. The immunologically active protein or peptide acts as an antigen and reacts with the antibodies which are present in the fluid under investigation. The test kits according to the invention can be based on various principles which are known per se. As a rule, a reaction takes place between the antigen and antibodies and this reaction, or the complex which is formed in this context, is detected. It is possible for the antigen to be bound to a solid phase such as a microtiter plate or magnetic beads. This antigen can then be brought into contact with the fluid under investigation (serum or cerebrospinal fluid). The antibodies which are present in the fluid under investigation then bind to the antigen. A wash is then customarily performed and the bound antibodies are detected by means of anti-antibody antibodies which carry a label. The label can be a radioactive isotope or an enzyme which catalyzes a color reaction, for example horseradish peroxidase.
However, there are a large number of test configurations which are known per se to the skilled person. Thus, the anti-antibody antibody can also, for example, be bound to a solid phase and the antigen can possess a detectable label.
Within the context of the present invention, preference is given, in particular, to those test kits which are suitable for implementing an ELISA (enzyme-linked immunosorbent assay) or for implementing a so-called Western blot.
Since the use of radioactively-labeled labeling substances is encountering ever increasing resistance, preference is given, according to the invention, to the complex, consisting of antigen/antibody to be detected and anti-antibody antibody, being detected by either the antigen or the anti-antibody antibody being biotinylated. The complex is then detected by adding avidin to which a color reaction-catalyzing enzyme, for example, is coupled.
Within the context of the present invention, particular preference is also given to the so-called xcexc capture test. In the xcexc capture test, an antibody against human IgM antibodies (xcexc chains) is bound to the solid phase. These anti-antibody antibodies capture both the antigen-specific and the nonspecific IgM antibodies from the serum mixture. After antigen, which can be directly labeled, has been added, the IgM immune response is detected. Alternatively, unlabeled antigen can also be employed, and the antigen (immunologically active protein according to the invention) is then detected using a further labeled antibody which is directed against the antigen. The label can, for example, be an enzyme which catalyzes a color reaction.
The immunologically active proteins or peptides according to the invention can also be used for preparing monoclonal antibodies. Monoclonal antibodies are prepared by means of standard methods which are known per se.
The present invention furthermore relates to vaccines which contain at least one protein or peptide according to the invention. Consequently, the immunologically active proteins, according to the invention, from Borrelia burgdorferi can be used for preparing a vaccine for protecting against infections with Borrelia burgdorferi bacteria.
For preparing a vaccine, it is essential to identify those regions in immunologically active proteins which elicit the formation of protective antibodies. When the immunologically active proteins are administered to the organism which is to be vaccinated, antibodies must be formed which are such that, in association with an infection with Borrelia burgdorferi, they bind to the invading bacteria and enable the invading bacteria to be destroyed by the body""s own immune system. While the vaccines according to the invention are preferably used for vaccinating humans, they can also be used for vaccinating animals. It is especially useful to vaccinate animals which can be bitten by ticks and thereby be infected with Borrelia burgdorferi. Vaccination is particularly useful in the case of dogs and horses.
The present invention also relates to nucleic acids which encode the immunologically active proteins according to the invention.
In this context, the nucleic acid is preferably a nucleic acid which exhibits a DNA sequence which encodes protein 1829-22A and possesses the sequence (SEQ ID NO:3).
ATGAAAAAGTTCAATTTAATAATTGAGGCGCTGTTTGCTATTCTATTAACAGCTTGTAATTTTGGATTAATGGAAGAAACAAAAATAGCGCTTGAATCATCCTCTAAGGATGTAAAAAATAAAATTTTACAAATAAAAAAAGACGCTGAGGACAAGGGTGTAAATTTTGCAGCTTTTACAAGCAGTGAAACCGGTTCTAAAGTGACAAATGGAGGATTAGCTTTAAGAGAAGCAAAAATACAAGCAATTAATGAAGTGGAAAAGTTTCTCAAGAGAATAGAAGAAGAGGCTTTAAAACTTAAAGAACATGGAAATAGTGGTCAATTCTTGGAGCTGTTTGACTTACTGCTTGAAGTTTTAGAATCATTAGAACCGATTGGAATAAAAGGCTTAAAAGACTTTATTTCAGAGGAAGCTAAATGTAACCCTATAAGCACATCTGAAAGATTAATTGAGGTTAAGGTGCAAATAGAAATAAGATGGAAGAGGTTAAGAGAAAACAAAATCTTAATAAGGAGAGAAAAAGTAATAAAGGCAAAAAAAAGAAATAA
or a part sequence thereof which encompasses at least 18 nucleotides.
In another embodiment, the nucleic acid is a nucleic acid which exhibits a DNA sequence which encodes protein 1829-22B and possesses the sequence (SEQ ID NO:4).
ATGATTAAATATAATAAAATTATACTTACACTAACTTTACTTGCTAGCCTGTTAGCAGCATGTAGTTTAACAGGAAAAGCTAGATTGGAATCATCAGTTAAAGACATTACAAATGAAATAGAGAAAGCTATAAAAGAAGCTGAAGACGCTGGTGTAAAGACAGACGCGTTCACAGAAACACAAACAGGTGGCAAGGTGGCAGGCCCTAAAATAAGAGCAGCAAAAATACGCGTCGCTGACTTAACAATCAAATTCCTAGAAGCAACAGAAGAGGAAACTATTACATTTAAAGAAAATGGAGCGGGGGAAGATGAATTCTCAGGAATATACGATTTAATACTCAACGCCGCAAAAGCAGTAGAAAAAATTGGGATGAAAGATATGACAAAAACGGTCGAAGAGGCCGCTAAAGAAAATCCTAAAACTACAGCTAATGGGATAATTGAGATTGTAAAAGTAATGAAAGCAAAAGTGGAAAACATTAAAGAAAAACAAACTAAAAATCAAAAATAA
or a part sequence thereof which encompasses at least 18 nucleotides.
According to the invention, preference is also given to part sequences of the abovementioned sequences, which part sequences possess at least 30 and particularly preferably 50 nucleotides.
The nucleic acids and nucleic acid fragments according to the invention, and nucleic acid fragments which hybridize with them and which have a length of at least 12 nucleotides, can be employed for detecting an infection with Borrelia burgdorferi using the polymerase chain reaction.
The nucleic acids according to the invention are preferably DNA sequences. The DNA sequences according to the invention are required for preparing the immunologically active proteins, according to the invention, from Borrelia burgdorferi by means of recombinant methods. However, it is also particularly advantageous to employ part sequences of the sequences according to the invention for diagnostic methods, with the PCR method having become very widespread. Short fragments of she nucleic acids according to the invention, which fragments are able to hybridize with the complementary sequences in the sample under investigation, are synthesized for this purpose. Very small quantities of the sought-after nucleic acids are then amplified by means of the polymerase chain reaction (PCR) and subsequently detected.
Another preferred use of the nucleic acids according to the invention is DNA vaccination. In this use, the nucleic acids according to the invention, or parts thereof, are introduced into the host to be immunized, in association with which the nucleic acid can either be present in naked form or in the form of plasmids or retroviral vectors. The DNA is then translated in the host organism and the translated gene products immunize the host.
The present invention is clarified by the following examples.